The colorimetric assay based on an enzyme reaction for signal amplification has seen wide use in the field of clinical diagnostics because it is convenient to use and has sufficient sensitivities. The sensitivity is dictated by the turn over rate of the enzyme label, the enzymatic substrates used and the amount of the enzyme. Alkaline phosphatase and horseradish peroxidase are two of the most popular enzyme labels. The former is often used in conjunction with nitro-blue tetrazolium chloride (NBT) and/or 5-bromo-4-chloro-3′-indolylphosphate p-toluidine salt (BCIP). The latter is often used in conjunction with 3,3′,5,5′-tetramethyl benzidine (TMB), 4-chloro-1-naphthol (4-CN), 3,3′-diaminobenzidine tetrahydrochloride (DAB) etc. Upon reaction of the enzymatic substrate with the enzyme, insoluble colored products precipitate. The insoluble product is detected visually for qualitative results, or with a simple optical detector for semi-quantitative results. Thus, how much insoluble product accumulates and how much optical density changes with accumulation dictates the detection limit.
Much effort has already gone into increasing the efficiency of the enzyme action and developing a substrate with an optically dense reaction product. There is not much room left for accelerating the enzymatic reaction by raising the temperature because higher temperatures lead to degradation of the enzyme. Increasing the amount of the enzyme does help, but there is a limit on how much antibody can be attached to a solid surface. This limit has already been reached with a porous membrane used for immuno-chromatographic kit; no drastic improvement can be expected. Another shortcoming associated with the colorimetric method is a poor dynamic range. As long as the detection method relied on measurement of absorbance, an accurate measurement is possible for optical densities below 2, and the minimum absorbance on the other hand is on the order of 0.1. Thus, the range is only 20, covering only slightly over one order of magnitude.
While improvements will certainly be made, there has not been a general approach applicable to a wide range of enzyme/substrate combinations. The present invention surprisingly offers a general approach that does not depend on improvements on a particular enzyme or substrate.